Cancer is generally a disease of the intracellular signalling system, or signal transduction mechanism. Normal cells respond to many extracellular sources, by proliferating or otherwise altering their metabolic activity. The signal transduction system receives such signals at the cell surface, and translates them into a message, decipherable by the cell for subsequent regulation of processes in cytoplasmic, nuclear, cytoskeletal, and membrane biochemistry. Cancer is commonly caused by a series of defects in these signalling proteins, resulting from a change either in their intrinsic activity or in their cellular concentrations. Frequently, these defects lead to a constituitive state whereby the cell nucleus receives an inappropriate signal to proliferate. This can occur through a variety of mechanisms. The cell may produce a growth factor that binds to its own receptors, resulting in an autocrine loop, which continually stimulates proliferation. Mutations can occur in the cell surface (tyrosine kinase) receptors, leading to activation of the kinase in the absence of ligand. Alternatively, many surface kinases can be overexpressed on the cell surface leading to an inappropriately strong response to a weak signal. Mutation or overexpression of many intracellular signalling proteins can lead to similar spurious mitogenic signals arising in the cell. Some of the most common of these mutations occur in the genes encoding the Ras protein, a G-protein which is activated when it is bound to GTP, and inactivated when it is bound to GDP.
The above mentioned growth factor receptors, and many other mitogenic receptors, when activated, lead to Ras being converted from the GDP-bound state to the GTP-bound state. This signal is an absolute prerequisite for proliferation in most cell types. Defects in this signalling system, especially in the deactivation of the Ras.GTP complex, are common in cancers, and lead to the signalling cascade below Ras being chronically activated.
Activated Ras leads in turn to the activation of a cascade of serine/threonine kinases. One of the groups of kinases known to require an active Ras.GTP for its own activation is the Raf family. These in turn activate Mek, which then activates MAP kinase. Activation of MAP kinase by mitogens appears to be essential for proliferation, and constituitive activation of this kinase is sufficient to induce cellular transformation. Blockade of downstream Ras signalling, for example by use of a dominant negative Raf-1 protein, can completely inhibit mitogenesis, whether induced from cell surface receptors or from oncogenic Ras mutants. Although Ras is not itself a protein kinase, it participates in the activation of Raf and other kinases, most likely through a phosphorylation mechanism. Once activated, Raf and other kinases phosphorylate MEK on two closely adjacent PG,4 serine residues, S.sup.218 and S.sup.222 in the case of MEK-1, which are the prerequisite for activation of MEK as a kinase. MEK in turn phosphorylates MAP kinase on both a tyrosine, Y.sup.185, and a threonine residue, T.sup.183, separated by a single amino acid. This double phosphorylation activates MAP kinase at least 100-fold, and it can now catalyze the phosphorylation of a large number of proteins, including several transcription factors and other kinases. Many of these MAP kinase phosphorylations are mitogenically activating for the target protein, whether it be another kinase, a transcription factor, or other cellular protein. MEK is also activated by several kinases other than Raf-1, including MEKK, and itself appears to be a signal integrating kinase. As far as is currently known, MEK is highly specific for the phosphorylation of MAP kinase. In fact, no substrate for MEK other than MAP kinase has been demonstrated to date, and MEK does not phosphorylate peptides based on the MAP kinase phosphorylation sequence, or even phosphorylate denatured MAP kinase. MEK also appears to associate strongly with MAP kinase prior to phosphorylating it, suggesting that phosphorylation of MAP kinase by MEK may require a prior strong interaction between the two proteins. Both this requirement and the unusual specificity of MEK are suggestive that it may have enough difference in its mechanism of action to other protein kinases that selective inhibitors of MEK, possibly operating through allosteric mechanisms rather than through the usual blockade of the ATP binding site, may be found
This invention provides a compound which is a highly specific inhibitor of the MEK kinase activity. Both in enzyme assays and whole cells, the compound inhibits the phosphorylation of MAP kinase by MEK, thus preventing the activation of MAP kinase in cells in which the Ras cascade has been activated. The results of this enzyme inhibition include a reversal of transformed phenotype of some cell types, as measured both by the ability of the transformed cells to grow in an anchorage-independent manner and by the ability of some transformed cell lines to proliferate independently of external mitogens.
The highly selective MEK inhibitory activity of the compound of the current invention is unexpected for several reasons. First, the compound is a member of the class of compounds known as the flavones, many of which are indiscriminate inhibitors of large numbers of protein kinases, so the degree of selectivity of this compound is unexpected. For example, Cushman, et al, J. Med. Chem. 1991;34:798-806, describe protein--tyrosine kinase inhibitory activities for a wide variety of flavonoids. Secondly, several close analogues of the invention compound are poor inhibitors of MEK. Lastly, no selective inhibitors of MEK have been reported to date. For example, Cunningham, et al, Anti-Cancer Drug Design 1992;7:365-84, Oxford University Press, 1992, describe the activity of several flavones, including a position isomer of the invention compound, which showed little selectivity.
Furthermore, the highly selective effect of the invention compound of the mitogenesis is unexpected. MAP kinase is activated by many cellular stimuli which are not mitogenic, and which mediate nonmitogenic cellular responses. For example, insulin treatment of adipocytes leads to upregulation of glucose transport and both glycogen and lipid synthesis, as well as the upregulation of MAP kinase activity, and it has been assumed that MAP kinase activation is required for the metabolic effects of insulin. The invention compound does inhibit activation of insulin stimulated MAP kinase, but it does not affect insulin-stimulated glucose transport or lipid and glycogen synthesis in murine 3T3-L1 adipocytes, demonstrating that the blockade that it causes can be selective for mitogenic effects over metabolic regulation.